SUMMARY Extracellular RNAs (exRNAs) have been found in all tested human biofluids, and there is increasing evidence that they can serve as mediators of intercellular communication, as well as diagnostic, prognostic, and theranostic biomarkers for a wide range of disease and physiological conditions. ExRNAs are associated with a variety of carriers subclasses (CSs), including extracellular vesicles (EVs), ribonucleoprotein complexes (RNPs), and lipoproteins (LPP), many of which are as-yet unknown or poorly characterized. This transdisciplinary team, with expertise in exRNA and lipoprotein biology, exRNA biomarker discovery, exRNA therapeutics, exRNA sequencing, low-input proteomics and lipidomics, and flow cytometry, will work together to develop and apply a rigorous, reproducible, efficient, scalable, and cost-effective immunomagnetic separation (IMS) workflow for preparative isolation of CSs for downstream omic analysis. In addition, the potential for multiplex bead-based flow sorting for even more efficient separation of CSs will be explored. Aim 1A will focus on development of reagents for identification and separation of known and suspected CSs using appropriate cell culture models and healthy human plasma and serum samples. This work will include screening of available antibodies against markers for known general CSs (e.g. tetraspanins, AGO proteins, apolipoproteins) and a variety of cell type- specific markers to identify antibodies that perform well for Western Blot and IMS, and dissemination of results for both successful and unsuccessful antibodies. In Aim 1B, the results from Aim 1A will be applied to build and test an IMS workflow for separation and small and long RNAseq, proteomic, and lipidomic analysis of general CSs and cell type-specific EVs. In addition to building a comprehensive knowledge set encompassing the RNA, protein, and lipid cargo of known and suspected CSs, profiling the material that is not captured by the IMS workflow will reveal novel CSs. Aim 2A will encompass refinement of the IMS workflow and application to three clinical cohorts: Pregnant and non-pregnant female controls; Post-myocardial infarction and age- and sex- matched at-risk controls; and Epithelial ovarian cancer and age-matched healthy female controls. Analysis of the exRNAs associated with general CSs and cell type-specific EVs by small and long RNAseq will reveal whether the relative abundance and exRNA cargo of of these CSs differs between cases and controls in these cohorts. Aim 2B will consist of development of a flow cytometry-based strategy for multiplexed simultaneous separation of multiple CSs from human plasma and serum. If successful, this project will result in development of a rigorous workflow for separation of exRNA CSs that reproducibly and rapidly produces fractions that are highly enriched for desired CSs with minimal contamination by other CSs in a cost-effective manner on clinically feasible volumes of input material, and yields sufficient material for downstream molecular analysis. In addition, the comprehensive omic data generated on during the course of this project will yield valuable reference profiling data on the RNA, protein, and lipid cargo carried by previously known and novel CSs.